Methods for producing transgenic plants

ABSTRACT

A method of producing a stably transformed corn plant in a single container is demonstrated. This method allows for the automation of the transformation process and reduces labor, material, and ergonomic costs associated with traditional plant tissue culture systems.

This application is a divisional of U.S. Ser. No. 13/945,727, filed Jul.18, 2013, which is a divisional of U.S. Ser. No. 13/355,312, filed Jan.20, 2012, now U.S. Pat. No. 8,513,016, issued Aug. 20, 2013, which is adivisional of U.S. Ser. No. 11/848,554, filed Aug. 31, 2007, now U.S.Pat. No. 8,124,411, issued Feb. 28, 2012, which application claimsbenefit of the priority of U.S. provisional application Ser. No.60/841,519 filed Aug. 31, 2006, each of the entire disclosures of whichare incorporated herein by reference.

BACKGROUND OF THE INVENTION

Field of the Invention

Micropropagation of plants has been done routinely in large batches andautomated. In micropropagation, an explant is usually taken up andplaced in one regeneration media that must be kept fresh for theduration of the regeneration process to produce a series of plants. Thisis in contrast to transformation processes, which are designed toproduce new transgenic events, and requires integration of foreign DNAinto a plant cell. Automating the plant tissue culture process,particularly the transformation process, has been difficult. The planttissues go through different stages that require is different kinds ofgrowth media and conditions. Transformation processes require multiplesteps and multiple media. For example, in Agrobacterium-mediatedtransformation, the process starts with the isolation of an explant thatis regenerable and transformable. Then the explant is inoculated withAgrobacterium in an inoculation media. After inoculation, excessAgrobacterium is typically removed and the explant and the Agrobacteriumare co-cultured together to allow the transfer of DNA. After co-culture,the presence of the Agrobacterium is deleterious to plant tissue culture(for example, causes unwanted contamination during subsequent handlingand tissue culture steps), so typically the explants are moved to freshmedium containing antibiotics to inhibit the growth of theAgrobacterium. This medium may or may not contain selection agents. Ifit does not, then it is called delay or resting medium. Explants may beplaced on delay medium to allow for some time to grow before optionallybeing placed on selection medium. Other protocols place the explantsdirectly into selection media for selection of transgenic events.Selection regimes vary widely depending upon the selection agent and theexplant system. Often multiple steps of selection are used and varyingamounts of selection agent can be necessary in the different steps.After selection of the transgenic events, the living transgenic eventsare then moved to regeneration media for regeneration to plantlets thatcan then be moved to soil. Up to the present time, transformationprocesses have been time-consuming and laborious and not able to be doneon a large scale. Automating transformation process would allow forlarge numbers of transgenic plants to be produced with reduced labor,material, and ergonomic burden.

The present invention has overcome the previous limitations intransformation by providing methods and apparatus that perform some orall of the transformation steps, and optionally some of the regenerationsteps, in a single container. Thus, the present methods overcome thedeficiencies of current transformation protocols by eliminatingtime-consuming steps required for sub-culturing plant tissue andchanging media. The methods and apparatus are particularly suitable fortransformation automation, regeneration automation, and/or large-scaleproduction of transformed cells, tissues, and plants.

The invention of genomics has enabled identification and isolation of alarge number of genes and has necessitated the need for reliable andefficient high throughput is transformation production systems fortesting the utility of these genes by transforming them intoeconomically important crops such as corn. Current corn transformationmethods requires, at least, four transfer steps from the step ofselecting a transformed cell to the step of transferring transgenicplants to soil thereby requiring higher material costs, for example,culture plates and media, and labor costs. Several manual transfers oftissues also elevate the risk of ergonomic injury due to repeatedmotions.

Thus, there is a need in the art of corn transformation for a highthroughput automated system for plant transformation, selection, andregeneration which can produce a large number of transgenic plants fortesting genes and creating useful plants while lowering material andlabor costs. There is also a need in the art for methods that can lowerrisk of ergonomic injuries making the work place safer.

Herein, the inventors provide a corn transformation method for selectingand regenerating transformed corn plants suitable for high throughputautomation system. The method employs a suitable support matrix incombination with liquid selection and regeneration medium. Use of thisliquid culture method eliminates the need for multiple transfers thatare normally required when using solid medium for selection andregeneration steps. Further, this method enables advance regenerationwhich has been a problem in liquid culture medium so far. Still further,the step of selecting a transformed cell and regeneration can beachieved in a single container such as a sundae cups until plants aretransferred to soil.

SUMMARY OF THE INVENTION

The present invention provides novel methods for automating planttransformation processes by providing a method of stably transforming,and optionally selecting and partially regenerating a plant in acontainer. In some embodiments, a single container may be used toperform the methods of the present invention. In other embodiments,multiple containers (for example, a system of interconnected vessels)may be provided for ease of use and optimization of the presentinvention.

In one aspect of the invention, method for producing a transgenic cornplant is provided. The method comprises obtaining a transformable cornexplant; transforming the transformable corn explant; selecting atransformed corn cell from the transformable corn explant on a selectionmedium; and regenerating the transformed cell into a plant on is aregeneration medium, wherein transforming, selecting, and regeneratingare done in the same container. Optionally, the selection in thecontainer may be omitted, and the transgenic plantlet or transgenicplant may be subject to selection after being placed into soil (forexample, sprayed with a selective agent).

The container may be a bioreactor, Petri plate, multi-well plate, flask,jar, bottle, jug, PlantCon™, temporary emersion system, and acombination thereof and is provided with a means for providing andremoving the medium. In some embodiments, the container is a Petriplate, multi-well plate, PlantCon, or temporary emersion system.

The explant may be selected from the group consisting of a callus,embryo, and a cell suspension.

The medium may be a liquid medium, solid medium, or a combinationthereof. In an embodiment, the selection medium is a solid medium andregeneration medium is a liquid medium overlaid over the solid selectionmedium.

The explant may be contacted with the medium temporarily. In anembodiment, the explant is contacted with the selection medium and theregeneration medium for about 1 to about 5 minutes about every 12 to 24hours.

In yet another aspect of the present invention, a method for obtainingexplant for producing a transgenic corn plant is provided. The methodcomprises dividing a callus into smaller callus pieces. In anembodiment, the callus is a type I callus.

In yet another aspect of the present invention, a method for preparingAgrobacterium cell suspension for inoculating an explant is provided.The method comprises culturing a frozen glycerol stock of Agrobacteriumdirectly into an induction medium.

In yet another aspect of the present invention, a corn cell culturecomprising transformable and embryogenic cells is provided.

In yet another aspect of the present invention, a method for producing acorn transformable and embryogenic cell culture is provided. The methodcomprises obtaining a callus from corn embryos and culturing the callusfor about 5 days to 30 days in a liquid medium to produce the cellculture. In an embodiment, the callus is a type II callus.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 Exemplary plasmid map of pMON30113.

DETAILED DESCRIPTION

The following definitions will aid in the understanding of thedescription of the invention.

“Callus” refers to a dedifferentiated proliferating mass of cells ortissue.

“Explant” refers to a plant part that is capable of being transformedand subsequently regenerated into a transgenic plant. Typical explantsinclude immature embryos, callus, cotyledons, meristems, leaves, orstems.

“Tissue culture media” refers to liquid, semi-solid, or solid media usedto support plant growth and development in a non-soil environment.Suitable plant tissue culture media is known to one of skill in the art,as discussed in detail subsequently. The media components can beobtained from suppliers other than those identified herein and can beoptimized for use by those of skill in the art according to theirrequirements.

“Coding sequence”, “coding region” or “open reading frame” refers to aregion of continuous sequential nucleic acid triplets encoding aprotein, polypeptide, or peptide sequence.

“Endogenous” refers to materials originating from within the organism orcell.

“Exogenous” refers to materials originating from outside of the organismor cell. It refers to nucleic acid molecules used in producingtransformed or transgenic host cells and plants. As used herein,exogenous is intended to refer to any nucleic acid that is introducedinto a recipient cell, regardless of whether a similar nucleic acid mayalready be present in such cell.

“Genome” refers to the chromosomal DNA of an organism. The genome isdefined as a haploid set of chromosomes of a diploid species. For thepurposes of this application, genome also includes the organellargenome.

“Monocot” or “monocotyledonous” refers to plants having a singlecotyledon. Examples include cereals such as maize, rice, wheat, oat, andbarley.

“Nucleic acid” refers to deoxyribonucleic acid (DNA) or ribonucleic acid(RNA).

“Phenotype” refers to a trait exhibited by an organism resulting fromthe interaction of genotype and environment.

“Polyadenylation signal” or “polyA signal” refers to a nucleic acidsequence located 3′ to a coding region that promotes the addition ofadenylate nucleotides to the 3′ end of the mRNA transcribed from thecoding region.

“Promoter” or “promoter region” refers to a nucleic acid sequence,usually found 5′ to a coding sequence, that controls expression of thecoding sequence by controlling production of messenger RNA (mRNA) byproviding the recognition site for RNA polymerase or other factorsnecessary for the start of transcription at the correct site.

“Recombinant nucleic acid vector” or “vector” refers to any agent suchas a plasmid, cosmid, virus, autonomously replicating sequence, phage,or linear or circular single- or double-stranded DNA or RNA nucleotidesegment, derived from any source, capable of genomic integration orautonomous replication, comprising a nucleic acid molecule in which oneor more nucleic acid sequences have been linked in a functionallyoperative manner. Such recombinant nucleic acid vectors or constructsare capable of introducing a 5′ regulatory sequence or promoter regionand a DNA sequence for a selected gene product into a cell in such amanner that the DNA sequence is transcribed into a functional mRNA,which is subsequently translated into a polypeptide or protein.

“Regeneration” refers to the process of growing a plant from a plantcell.

“Regeneration medium” refers to a plant tissue culture medium requiredfor containing a selection agent.

“Regenerable callus” refers to callus from which whole plants can beproduced but where the mode of regeneration (embryogenesis ororganogenesis) has not been determined or is not pertinent to thediscussion.

“Selectable marker” or “screenable marker” refers to a nucleic acidsequence whose expression confers a phenotype facilitatingidentification of cells containing the nucleic acid sequence.

“Selection” refers to contacting an inoculated explant with a selectionmedium for obtaining a transformed cell, tissue, or plant.

“Selection medium” refers to a plant tissue culture medium containing aselection is agent.

“Transcription” refers to the process of producing an RNA copy from aDNA template.

“Transformation” refers to a process of introducing an exogenous nucleicacid sequence (vector or construct) into a cell or protoplast, in whichthat exogenous nucleic acid is incorporated into the nuclear DNA,plastid DNA, or is capable of autonomous replication.

“Transgenic” refers to organisms into which an exogenous nucleic acidsequence has been integrated.

“Transformable explant” refers to any part of a plant that is receptiveto transformation.

The present invention provides a system in which stable transformationcan be carried out in a single container. The transformation processbegins with inoculation of the transformable explant with Agrobacteriumand results in a stably transformed plantlet with roots suitable fortransfer to soil.

There are many containers that can be used for this purpose.Bioreactors, including the temporary immersion system, can be used. Manydifferent containers have been used for plant liquid tissue culture,including, but not limited to, Petri plates of various sizes, multi-wellplates, flasks, jars, bottles, jugs, and PlantCons. These containers areusually provided with means such as an inlet and outlet for providingthe fresh medium and removing the expensed medium. A plurality ofcontainers may be connected to obtain a high-throughput system.

These containers may include some support for the explant. That supportcan be, but is not limited to, filter paper, felt, rafts, glass beads,zirconia/silica beads, foam, or solid media. Liquid media is usuallyplaced in the container and then exchanged as needed. This exchange canbe done manually or mechanically.

The containers may contain many explants at a time or may be smallenough to contain a single explant. In the case of multi-well plates, anarray of small wells each containing an explant is used to culture largenumbers of explants. Advantages of the multi-well plates include theisolation of any contaminated explants. The explants may be preparedmanually or mechanically.

The purpose of the invention is to have a system that is easilyautomatable from start to finish; however, any of these liquid culturesystems and containers can be used in combination with othertransformation, selection, and regeneration steps known to one of skillin the art.

A high throughput transformation system can be developed in whichcontainers may be manipulated by robotic arms on a freely configurablework table that may include incubators and shakers in addition tostandard lab ware. Various liquid handling tools equipped with one ormore pipetting tips can be used to provide the fresh medium and removethe expensed medium. Work table, robotic arms, and the liquid handlingtools can be controlled by software via a computer. Alternatively,liquid medium for selecting and regenerating the transformed cell can beprovided to the container via one or more tube(s) connected to a mediumstorage vessel and removed via one or more tubes connected to a wastevessel. The provision and removal of the medium can be controlledmanually or mechanically.

To initiate a transformation process in accordance with the presentinvention, it is first necessary to select genetic components to beinserted into the plant cells or tissues. Genetic components can includeany nucleic acid that is introduced into a plant cell or tissue usingthe method according to the invention. Genetic components can includenon-plant DNA, plant DNA or synthetic DNA.

In a preferred embodiment, the genetic components are incorporated intoa DNA composition such as a recombinant, double-stranded plasmid orvector molecule comprising at least one or more of following types ofgenetic components: (a) a promoter that functions in plant cells tocause the production of an RNA sequence, (b) a structural DNA sequencethat causes the production of an RNA sequence that encodes a product ofagronomic utility, and (c) a 3′ non-translated DNA sequence thatfunctions in plant cells to cause the addition of polyadenylatednucleotides to the 3′ end of the RNA sequence.

The vector may contain a number of genetic components to facilitatetransformation of the plant cell or tissue and regulate expression ofthe desired gene(s). In one preferred embodiment, the genetic componentsare oriented so as to express an mRNA, which in one embodiment can betranslated into a protein. The expression of a plant structural codingsequence (a gene, cDNA, synthetic DNA, or other DNA) that is exists indouble-stranded form involves transcription of messenger RNA (mRNA) fromone strand of the DNA by RNA polymerase enzyme and subsequent processingof the mRNA primary transcript inside the nucleus. This processinginvolves a 3′ non-translated region that adds polyadenylated nucleotidesto the 3′ ends of the mRNA.

Means for preparing plasmids or vectors containing the desired geneticcomponents are well known in the art. Vectors typically consist of anumber of genetic components, including but not limited to regulatoryelements such as promoters, leaders, introns, and terminator sequences.Regulatory elements are also referred to as cis- or trans-regulatoryelements, depending on the proximity of the element to the sequences orgene(s) they control.

Transcription of DNA into mRNA is regulated by a region of DNA usuallyreferred to as the “promoter”. The promoter region contains a sequenceof bases that signals RNA polymerase to associate with the DNA and toinitiate the transcription into mRNA using one of the DNA strands as atemplate to make a corresponding complementary strand of RNA.

A number of promoters that are active in plant cells have been describedin the literature. Such promoters would include but are not limited tothe nopaline synthase (NOS) and octopine synthase (OCS) promoters thatare carried on tumor-inducing plasmids of Agrobacterium tumefaciens, thecaulimovirus promoters such as the cauliflower mosaic virus (CaMV) 19Sand 35S promoters and the figwort mosaic virus (FMV) 35S promoter, theenhanced CaMV35S promoter (e35S), the light-inducible promoter from thesmall subunit of ribulose bisphosphate carboxylase (ssRUBISCO, a veryabundant plant polypeptide). All of these promoters have been used tocreate various types of DNA constructs that have been expressed inplants.

Promoter hybrids can also be constructed to enhance transcriptionalactivity (U.S. Pat. No. 5,106,739), or to combine desiredtranscriptional activity, inducibility and tissue specificity ordevelopmental specificity. Promoters that function in plants include butare not limited to promoters that are inducible, viral, synthetic,constitutive as described, and temporally regulated, spatiallyregulated, and spatio-temporally regulated. Other promoters that aretissue-enhanced, tissue-specific, or developmentally regulated are alsoknown in the art and envisioned to have utility in the practice of thisinvention.

Promoters may be obtained from a variety of sources such as plants andplant DNA viruses and include, but are not limited to, the CaMV35S andFMV35S promoters and promoters isolated from plant genes such asssRUBISCO genes. As described below, it is preferred that the particularpromoter selected should be capable of causing sufficient expression toresult in the production of an effective amount of the gene product ofinterest.

The promoters used in the DNA constructs (for example,chimeric/recombinant plant genes) of the present invention may bemodified, if desired, to affect their control characteristics. Promoterscan be derived by means of ligation with operator regions, random orcontrolled mutagenesis, etc. Furthermore, the promoters may be alteredto contain multiple “enhancer sequences” to assist in elevating geneexpression.

The mRNA produced by a DNA construct of the present invention may alsocontain a 5′ non-translated leader sequence. This sequence can bederived from the promoter selected to express the gene and can bespecifically modified so as to increase translation of the mRNA. The 5′non-translated regions can also be obtained from viral RNAs, fromsuitable eukaryotic genes, or from a synthetic gene sequence. Such“enhancer” sequences may be desirable to increase or alter thetranslational efficiency of the resultant mRNA. The present invention isnot limited to constructs wherein the non-translated region is derivedfrom both the 5′ non-translated sequence that accompanies the promotersequence. Rather, the non-translated leader sequence can be derived fromunrelated promoters or genes (see, for example U.S. Pat. No. 5,362,865).Other genetic components that serve to enhance expression or affecttranscription or translation of a gene are also envisioned as geneticcomponents.

The 3′ non-translated region of the chimeric constructs should contain atranscriptional terminator, or an element having equivalent function,and a polyadenylation signal that functions in plants to cause theaddition of polyadenylated nucleotides to the 3′ end of the RNA.Examples of suitable 3′ regions are (1) the 3′ transcribed,non-translated regions containing the polyadenylation signal ofAgrobacterium tumor-inducing (Ti) plasmid genes, such as the nopalinesynthase (NOS) gene, and (2) plant genes such as the soybean storageprotein genes and the small subunit of the ribulose-1,5-bisphosphatecarboxylase (ssRUBISCO) gene. An example of a is preferred 3′ region isthat from the ssRUBISCO E9 gene from pea (European Patent Application0385 962).

Typically, DNA sequences located a few hundred base pairs downstream ofthe polyadenylation site serve to terminate transcription. The DNAsequences are referred to herein as transcription-termination regions.The regions are required for efficient polyadenylation of transcribedmessenger RNA (mRNA) and are known as 3′ non-translated regions. RNApolymerase transcribes a coding DNA sequence through a site wherepolyadenylation occurs.

In one preferred embodiment, the vector contains a selectable,screenable, or scoreable marker gene. These genetic components are alsoreferred to herein as functional genetic components, as they produce aproduct that serves a function in the identification of a transformedplant, or a product of agronomic utility. The DNA that serves as aselection device functions in a regenerable plant tissue to produce acompound that would confer upon the plant tissue resistance to anotherwise toxic compound. Genes of interest for use as a selectable,screenable, or scorable marker would include but are not limited to GUS,green fluorescent protein (GFP), anthocyanin biosynthesis related genes(C1, Bperu), luciferase (LUX), antibiotics like kanamycin (Dekeyser etal., 1989), and herbicides like glyphosate (Della-Cioppa et al., 1987).Other selection devices can also be implemented including but notlimited to tolerance to phosphinothricin, bialaphos, dicamba, andpositive selection mechanisms and would still fall within the scope ofthe present invention.

The present invention can be used with any suitable plant transformationplasmid or vector containing a selectable or screenable marker andassociated regulatory elements as described, along with one or morenucleic acids expressed in a manner sufficient to confer a particulartrait. Examples of suitable structural genes of agronomic interestenvisioned by the present invention would include but are not limited togenes for insect or pest tolerance, herbicide tolerance, genes forquality improvements such as yield, nutritional enhancements,environmental or stress tolerances, or any desirable changes in plantphysiology, growth, development, morphology or plant product(s).

Alternatively, the DNA coding sequences can affect these phenotypes byencoding a non-translatable RNA molecule that causes the targetedinhibition of is expression of an endogenous gene, for example viaantisense- or cosuppression-mediated mechanisms (see for example, Birdet al., 1991). The RNA could also be a catalytic RNA molecule (forexample, a ribozyme) engineered to cleave a desired endogenous mRNAproduct (see for example, Gibson and Shillitoe, 1997). Moreparticularly, for a description of anti-sense regulation of geneexpression in plant cells see U.S. Pat. No. 5,107,065 and for adescription of gene suppression in plants by transcription of a dsRNAsee U.S. Pat. No. 6,506,559, U.S. Patent Application Publication No.2002/0168707 A1, and U.S. patent application Ser. No. 09/423,143 (see WO98/53083), 09/127,735 (see WO 99/53050) and 09/084,942 (see WO99/61631), all of which are incorporated herein by reference. Thus, anygene that produces a protein or mRNA that expresses a phenotype ormorphology change of interest is useful for the practice of the presentinvention.

Exemplary nucleic acids that may be introduced by the methodsencompassed by the present invention include, for example, DNA sequencesor genes from another species, or even genes or sequences that originatewith or are present in the same species, but are incorporated intorecipient cells by genetic engineering methods rather than classicalreproduction or breeding techniques. However, the term exogenous is alsointended to refer to genes that are not normally present in the cellbeing transformed, or perhaps simply not present in the form, structure,etc., as found in the transforming DNA segment or gene, or genes thatare normally present yet that one desires, for example, to haveover-expressed. Thus, the term “exogenous” gene or DNA is intended torefer to any gene or DNA segment that is introduced into a recipientcell, regardless of whether a similar gene may already be present insuch a cell. The type of DNA included in the exogenous DNA can includeDNA that is already present in the plant cell, DNA from another plant,DNA from a different organism, or a DNA generated externally, such as aDNA sequence containing an antisense message of a gene, or a DNAsequence encoding a synthetic or modified version of a gene.

In light of this disclosure, numerous other possible selectable orscreenable marker genes, regulatory elements, and other sequences ofinterest will be apparent to those of skill in the art. Therefore, theforegoing discussion is intended to be exemplary rather than exhaustive.

The technologies for the introduction of DNA into cells are well knownto those of skill in the art and can be divided into categoriesincluding but not limited to: (1) chemical methods; (2) physical methodssuch as microinjection, electroporation, and micro-projectilebombardment; (3) viral vectors; (4) receptor-mediated mechanisms; and 5)Agrobacterium-mediated plant transformation methods.

For Agrobacterium-mediated transformation, after the construction of theplant transformation vector or construct, said nucleic acid molecule,prepared as a DNA composition in vitro, is introduced into a suitablehost such as E. coli and mated into another suitable host such asAgrobacterium, or directly transformed into competent Agrobacterium.These techniques are well-known to those of skill in the art and havebeen described for a number of plant systems including soybean, cotton,and wheat (see, for example U.S. Pat. Nos. 5,569,834 and 5,159,135, andWO 97/48814, herein incorporated by reference in their entirety).

The present invention encompasses the use of bacterial strains tointroduce one or more genetic components into plants. Those of skill inthe art would recognize the utility of Agrobacterium-mediatedtransformation methods. A number of wild-type and disarmed strains ofAgrobacterium tumefaciens and Agrobacterium rhizogenes harboring Ti orRi plasmids can be used for gene transfer into plants. Preferably, theAgrobacterium hosts contain disarmed Ti and Ri plasmids that do notcontain the oncogenes that cause tumorigenesis or rhizogenesis,respectively, which are used as the vectors and contain the genes ofinterest that are subsequently introduced into plants. Preferred strainswould include but are not limited to disarmed Agrobacterium tumefaciensstrain C58, a nopaline-type strain that is used to mediate the transferof DNA into a plant cell, octopine-type strains such as LBA4404 orsuccinamopine-type strains, for example, EHA101 or EHA105. Otherbacteria such as Sinorhizobium, Rhizobium, and Mesorhizobium thatinteract with plants naturally can be modified to mediate gene transferto a number of diverse plants. These plant-associated symbiotic bacteriacan be made competent for gene transfer by acquisition of both adisarmed Ti plasmid and a suitable binary vector (Broothaerts et al,2005).

The use of these strains for plant transformation has been reported andthe methods are familiar to those of skill in the art.

The explants can be from a single genotype or from a combination ofgenotypes. Any corn seed that can germinate is a viable startingmaterial. In a preferred embodiment, superior explants from planthybrids can be used as explants. For example, a fast-growing cell linewith a high culture response (higher frequency of embryogenic callusformation, growth rate, plant regeneration frequency, etc.) can begenerated using hybrid embryos containing several genotypes. In apreferred embodiment, an F1 hybrid or first generation offspring ofcross-breeding can be used as a donor plant and crossed with anothergenotype. Those of skill in the art are aware that heterosis, alsoreferred to herein as “hybrid vigor”, occurs when two inbreds arecrossed. The present invention thus encompasses the use of an explantresulting from a three-way cross, wherein at least one or more of theinbreds is highly regenerable and transformable, and the transformationand regeneration frequency of the three-way cross explant exceeds thefrequencies of the inbreds individually. Other tissues are alsoenvisioned to have utility in the practice of the present invention.Explants can include mature embryos, immature embryos, meristems, callustissue, or any other tissue that is transformable and regenerable.

Any suitable plant culture medium can be used during the transformationprocess. Examples of suitable media would include but are not limited toMS-based media (Murashige and Skoog, 1962) or N6-based media (Chu etal., 1975) supplemented with additional plant growth regulatorsincluding but not limited to auxins such as picloram(4-amino-3,5,6-trichloropicolinic acid), 2,4-D(2,4-dichlorophenoxyacetic acid) and dicamba (3,6-dichloroanisic acid);cytokinins such as BAP (6-benzylaminopurine) and kinetin; ABA; andgibberellins;. Other media additives can include but are not limited toamino acids, macroelements, iron, microelements, inositol, vitamins andorganics, carbohydrates, undefined media components such as caseinhydrolysates, ethylene antagonists including silver nitrate with orwithout an appropriate gelling agent such as a form of agar, such as alow melting point agarose or Gelrite® if desired. Those of skill in theart are familiar with the variety of tissue culture media, which whensupplemented appropriately, support plant tissue growth and developmentand are suitable for plant transformation and regeneration. These tissueculture media can either be purchased as a is commercial preparation orcustom prepared and modified. Examples of such media would include butare not limited to Murashige and Skoog (1962), N6 (Chu et al., 1975),Linsmaier and Skoog (1965), Uchimiya and Murashige (1962), Gamborg'smedia (Gamborg et al., 1968), D medium (Duncan et al., 1985), McCown'sWoody plant media (McCown and Lloyd, 1981), Nitsch and Nitsch (1969),and Schenk and Hildebrandt (1972) or derivations of these mediasupplemented accordingly. Those of skill in the art are aware that mediaand media supplements such as nutrients and growth regulators for use intransformation and regeneration and other culture conditions such aslight intensity during incubation, pH, and incubation temperatures canbe optimized for the particular variety of interest.

Once the transformable plant tissue is isolated, the next step of themethod is introducing the genetic components into the plant tissue. Thisprocess is also referred to herein as “transformation.” The plant cellsare transformed and each independently transformed plant cell isselected. The independent transformants are referred to as transgenicevents. A number of methods have been reported and can be used to insertgenetic components into transformable plant tissue. Micro-projectilebombardment and Agrobacterium-mediated gene delivery are the two mostcommonly used plant transformation methods, but other methods are known.

Those of skill in the art are aware of the typical steps in the planttransformation process. Those of skill in the art are familiar withprocedures for growth and suitable culture conditions for Agrobacteriumas well as subsequent inoculation procedures. The density of theAgrobacterium culture used for inoculation and the ratio ofAgrobacterium cells to explant can vary from one system to the next, andtherefore optimization of these parameters for any transformation methodis expected. Agrobacterium can also be induced directly from frozenstocks or can be cultured in multiple ways known to one of skill in theart.

The next stage of the transformation process is the inoculation. In thisstage the explants and Agrobacterium cell suspensions are mixedtogether. This can be achieved by incubating explants in Agrobacteriumcell suspension. In other embodiments described herein, the explantswere inoculated while they were still attached to the donor tissue,while removing the explants from the donor tissue, after plating theexplants onto the selection medium, or by a combination. The durationand condition of the inoculation and Agrobacterium cell density willvary depending on the plant transformation system.

After inoculation, any excess Agrobacterium suspension can be removedand the Agrobacterium and target plant material are co-cultured. Theco-culture refers to the time post-inoculation and prior to transfer toa delay or selection medium. Any number of plant tissue culture mediacan be used for the co-culture step. Plant tissues after inoculationwith Agrobacterium can be cultured in a liquid or a semi-solid media.The co-culture is typically performed for about one to three days at atemperature of about 18° C.-30° C. The co-culture can be performed inthe light or in light-limiting conditions. Lighting conditions can beoptimized for each plant system as is known to those of skill in theart.

After co-culture with Agrobacterium, the explants typically can beplaced directly onto selective media. Alternatively, after co-culturewith Agrobacterium, the explants could be placed on media without theselective agent and subsequently placed onto selective media. Those ofskill in the art are aware of the numerous modifications in selectiveregimes, media, and growth conditions that can be varied depending onthe plant system and the selective agent. Typical selective agentsinclude but are not limited to antibiotics such as geneticin (G418),kanamycin, paromomycin or other chemicals such as glyphosate,phosphinothricin, bialaphos, and dicamba. Additional appropriate mediacomponents can be added to the selection or delay medium to inhibitAgrobacterium growth. Such media components can include, but are notlimited to, antibiotics such as carbenicillin or cefotaxime.

In one embodiment, inoculation, co-culture and selection steps werecombined into a single step by plating the inoculated explants directlyonto a medium that contained selective agents for suppressing growth ofAgrobacterium and killing non-transformed explant cells for improvingtransformation production system efficiency.

The cultures are subsequently transferred to a media suitable for therecovery of transformed plantlets. Those of skill in the art are awareof the number of methods to recover transformed plants. A variety ofmedia and transfer requirements can be implemented and optimized foreach plant system for plant transformation and recovery is of transgenicplants. Consequently, such media and culture conditions disclosed in thepresent invention can be modified or substituted with nutritionallyequivalent components, or similar processes for selection and recoveryof transgenic events, and still fall within the scope of the presentinvention.

The present invention includes all of the previously described steps;however, modifications are made as appropriate to facilitate the processin a single container. Liquid culture on various types of support isused to facilitate changing the media from step to step. A temporaryimmersion system bioreactor or other device giving similar results canbe used for media replacement.

In the case of callus as the explant, the callus can be minced usingvarious devices including but not limited to a garlic press, scissors,scalpels, or other cutting devices. The callus can be minced very fineto fit into a small multi-well plate system, where the expectation is toobtain a single event in each well. These modifications provideergonomic relief and can be used to recover many transgenic events froma single piece of callus.

The transformants produced are subsequently analyzed to determine thepresence or absence of a particular nucleic acid of interest containedon the transformation vector. Molecular analyses can include but are notlimited to Southern blots (Southern, 1975), or PCR (polymerase chainreaction) analyses, immunodiagnostic approaches, and field evaluations.These and other well known methods can be performed to confirm thestability of the transformed plants produced by the methods disclosed.These methods are well known to those of skill in the art and have beenreported (see for example, Sambrook et al., 1989).

Those of skill in the art will appreciate the many advantages of themethods and compositions provided by the present invention. Thefollowing examples are included to demonstrate the preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples that follow representtechniques discovered by the inventors to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments that are disclosed and still obtain alike or is similar result without departing from the spirit and scope ofthe invention. All references cited herein are incorporated herein byreference to the extent that they supplement, explain, provide abackground for, or teach methodology, techniques, or compositionsemployed herein.

EXAMPLES

The following examples are given for the purpose of illustrating variousembodiments of the invention and are not meant to limit the presentinvention in any fashion. One skilled in the art will appreciate readilythat the present invention is well adapted to carry out the objects andobtain the ends and advantages mentioned, as well as those objects, endsand advantages inherent herein. The present examples, along with themethods described herein are presently representative of preferredembodiments, are exemplary, and are not intended as limitations on thescope of the invention. Changes therein and other uses which areencompassed within the spirit of the invention as defined by the scopeof the claims will occur to those skilled in the art.

Example 1 Explant Source and Culture Conditions

Eight-day-old callus (eight days post sub-culture) obtained fromimmature embryos of corn was cultured on gelled 211V medium (N6 basalsalt mixture, 1 mg/L 2-4-D, 1 mg/L Thiamine HCL, 1 mg/L Nicotinic Acid,0.91 g/L L-Asparagine Monohydrate, 0.1 g/L myo-inositol, 0.5 g/L MES,1.6 g/L MgCl₂.6H₂O, 0.1 g/L casein hydrolysate, 0.69 g/L Proline, 20 g/Lsucrose, 0.1 mM silver nitrate, 6 g/L Phyta agar).

Wherever gelled medium use is mentioned, 25×100 mm Petri platescontaining 50 mL of medium with 12 explants per plate were used and alltransfers were done manually.

Liquid medium consisted of 212V (N6 basal salts with 2 mg/L 2-4-D, 1mg/L Thiamine HCL, 1 mg/L Nicotinic Acid, 0.91 g/L L-AsparagineMonohydrate, 0.1 g/L myo-inositol, 0.5 g/L MES, 1.6 g/L MgCl₂.6H₂O, 0.1g/L casein hydrolysate, 0.69 g/L Proline, 20 g/L sucrose, 0.01 mM silvernitrate, pH adjusted to 5.8 and autoclaved at 121° C. and 105 kPa for 20min. Post autoclave, filter sterilized Carbenicillin and Paromomycin at250 mg/L or 500 mg/L and 100 mg/L or 200 mg/L of concentration was addedto the medium as a selective agent.

For liquid culture, either a Temporary Immersion System (TIS) vessel,240×240 mm large Petri-plates, or 25×100 mm Petri-plates, or multi-wellplates were used. All cultures were incubated at 27-28° C. in the darkin a growth cabinet (Percival scientific Series 101 US).

Agrobacterium tumefaciens infection and co-cultivation. Activelygrowing, uniform sized, eight-day-old callus was selected and inoculatedwith Agrobacterium suspension (0.25×10⁹ cfu/mL) for 30 minutes. Thecalli were washed with 212V liquid medium containing 0.1 mM AgNO₃ andtransferred to TIS container or 25×100 mm Petri plate containing filterpaper or large shallow Petri-plate (240×240 mm) containing two layers of(220×220 mm) polyester felt. Co-cultivation treatments were carried outin the dark at 23° C.

A. tumefaciens strains and plasmids. ABI Agrobacterium strain C58 wasused to mediate the transfer of DNA into the plant cells. The plasmidpMON30113 (FIG. 1) contained neomycin phosphotransferase II gene (NPTII)as the selectable marker and a green fluorescence protein gene (GFP) asthe screenable marker driven by the 35S promoter.

Example 2 Temporary Immersion System (TIS)

A modified TIS was constructed as follows. Two chambers of anautoclavable 250 mL filter unit (Nalge Nunc Intl., Rochester, N.Y.) wereconnected by insertion of a glass tube into the base of the supportplate normally used to hold the membrane filter. Air hoses wereconnected to the two chambers. The airflow was sterilized by passagethrough 0.2 μm pore size, 50 mm diameter hydrophobic PTFE membranefilters (Millipore, Billerica, Mass.). Liquid medium was placed in thelower chamber and explant materials were placed on a supporting disc inthe upper chamber. Air was pumped into the lower chamber to displace theliquid medium through the glass tube to the upper chamber, immersing thecallus. Air pumped to the upper chamber forced the medium to return tothe lower chamber (only a thin film of liquid medium remains on thetissues). An electronic timer was used to operate solenoids that controlairflow to either the upper or lower chamber. The timer was programmedfor either airflow into the lower chamber, airflow to the upper chamberor “idle time” (no airflow). Airflow times were set at 1 to 5 min eachwhile the idle time could vary. The program was then set to run in acontinuous cycle through the different stages of the transformationprocess. In this way, tissues were completely immersed for approximately1-5 min at set time intervals. This is called immersion cycle frequency.

Optimization of immersion cycle frequencies. Tissue was cultured at 1min duration submersion and four different immersion cycle frequencies(once every 6 h, 8 h, 12 h and 24 h) each, for a period of 8 weeks weretested. As a control, half of the calli derived from the same ear werecultured on 211V gelled medium with selection.

The immersion time interval was found to significantly impact theproliferation rate of callus in the TIS. The maximum rate of calliproliferation and biomass increase occurred using an immersion frequencyof either 12 h or 24 h. With these two immersion cycle frequencies,70-80% of calli produced GFP positive sectors. By increasing theimmersion frequency to one cycle every 6 or 8 hours, no sign of callusproliferation was observed and within 10 to 14 days, callus turned brownand eventually died.

Effect of Carbenicillin on A. tumefaciens Growth. Calli were inoculatedwith pMON30113 as described above. Following inoculation, the infectedcalli were transferred to callus proliferation medium (212V)supplemented with either 0, 100, 250, 500 or 1000 mg/L carbenicillin.Growth of Agrobacterium was visually evaluated by observing turbidity ofthe liquid medium in the lower chamber of the TIS apparatus. A separateTIS unit with non-inoculated control treatment was included. Anadditional control of non-inoculated callus was also cultured on gelledmedium supplemented with carbenicillin (211V) to study the effect ofcarbenicillin on callus culture.

No significant difference in callus quality was noticed whennon-inoculated callus cultured on 211V (gelled) and 212V (liquid) mediumsupplemented with or without carbenicillin, indicating that there is nonegative effect of carbenicillin on growth and development ofnon-inoculated callus. However, differences in growth of A. tumefaciensand GFP-positive sectors were observed as the concentration ofcarbenicillin increased from 0.0 to 1000 mg/L. Carbenicillin at aconcentration of 100 mg/L could not suppress is the growth ofAgrobacterium either in liquid or gelled medium. The optimumconcentration of carbenicillin was found to be 500 mg/L for gelled andliquid medium

Effect of Paromomycin on Calli and Shoot Proliferation. To inhibitnon-transgenic callus proliferation, both gelled (control) and liquidculture medium were supplemented with the selective agent paromomycin(25, 50, 75, 100, or 200 mg/L). Control treatments were (1)non-inoculated callus on selective medium, (2) non-inoculated callus onselection-free medium, and (3) inoculated callus on selection-freemedium. The basic culture medium consisted of either 211V or 212V. Forthis experiment 25×100 mm deep Petri-plates were used. With liquidculture, two layers of 60×60 mm felt were used to hold the explants. Theselective agent for Agrobacterium-inoculated culture containedcarbenicillin and paromomycin.

Paromomycin at low concentrations more effectively inhibited embryogeniccallus growth and plant regeneration in liquid medium than the gelledmedium. No plants were obtained from non-inoculated callus from liquidmedium containing as low as 50 mg/L of paromomycin whereas 50% of thecallus produced plantlets on gelled medium at the same concentration ofparomomycin. Paromomycin at a concentration of 100 mg/L inhibited callusgrowth with both liquid and gelled medium.

Optimization of Amount of Liquid Medium Used in TIS. Developing calluswas cultured in one of four different amounts of liquid medium (20 mL,30 mL, 50 mL and 100 mL/container).

Selection and Regeneration in TIS. After 3 days of co-culture in TIS, 20mL of selection medium 212VRT (N6 basal salts with 2 mg/L 2,4-D, 1 mg/LThiamine HCL, 1 mg/L Nicotinic Acid, 0.91 g/L L-Asparagine Monohydrate,0.1 g/L myo-inositol, 0.5 g/L MES, 1.6 g/L MgCl₂.6H₂O, 0.1 g/L caseinhydrolysate, 0.69 g/L Proline, 20 g/L sucrose, 0.01 mM silver nitrate,500 mg/L Carbenicillin and 100 mg/L Paromomycin) was injected in lowerchamber of TIS. Calli were immersed for one minute once every 12 hours.After two weeks of incubation, 212VRT medium was aspirated from thelower chamber using a vacuum pump and replaced with 20 mL of 212RT (nosilver nitrate) medium. Calli were immersed for one minute once every 24hours for two more weeks. After completing 4 weeks of selection phase,selection medium was replaced with BAP-pulse medium 217A (N6 basal saltswith 1 mg/L Thiamine HCL, 1 mg/L Nicotinic Acid, 3.52 mg/L BAP, 0.91 g/LL-Asparagine Monohydrate, 0.1 g/L myo-inositol, 0.5 g/L MES, 1.6 g/LMgCl₂.6H₂O, 0.1 g/L casein hydrolysate, 0.69 g/L Proline, 20 g/Lsucrose, 250 mg/L Carbenicillin). Calli were treated with 217A mediumfor 7 days with one minute of immersion once every 12 hours.

For plant regeneration, 217A medium was replaced with 632AG medium (MSsalts mixture, MS vitamins, 50 mg/L myo-inositol, sucrose 60 g/L,paromomycin 50 mg/L and carbenicillin 250 mg/L). Medium was injectedinto the upper chamber of TIS and two quick runs of one minute ofimmersion were performed to dilute out any residual 217A medium. Lightsin the Percival were turned on to a 16 h on/8 h off cycle. TIS was leftidle (without any immersion) for 72 hours and thereafter calli wereimmersed once every 24 hours till shoots developed to the size of 5-20mm.

After 10-12 weeks of selection and regeneration in one vessel,transgenic plants were obtained. A transformation frequency of 5.9% wasachieved in the TIS. This compares to 17.7% in the control.

Example 3 Use of Petri Plates

The calli obtained as in Example 1 were washed with 212V liquid mediumcontaining 0.1 mM AgNO₃ and transferred to either a 25×100 mm Petriplate containing filter paper or large shallow Petri-plate (240×240 mm)containing two layers of (220×220 mm) acrylic felt.

Explants were then cultured as described in the TIS in Example 2 withthe difference being that the liquid media exchanges were done by hand.The liquid media was compared with gelled media. In the smaller Petriplate, liquid media gave a transformation frequency of 4.2% compared to6.6% with gelled media. In the larger Petri plate, liquid media gave atransformation frequency of 7.8% compared to 11.5% with gelled media.This demonstrates that liquid media is a viable alternative to thestandard solid media protocols. Liquid media will allow for greaterautomation.

Example 4 Use of Multiple-Well Plates as Container

Multi-well plates can also be used as the single container system.Experiments were done as described above except for the use ofmulti-well plates instead of Petri plates. Multi-well plates from Costar(Corning Life Sciences, Acton, Mass.) were used with or without theirTranswell™ inserts. The size of the membrane pores used (0.1μ) allowsthe media to seep across the membrane to the corn tissue as needed,without flooding the tissue. Pipette tip access ports allow media to bequickly removed and added into the wells for media changes. During mediatransfers, the media within the wells is changed by aspirating out theold media with a vacuum hose and pipetting in new media.

The multi-well plates used in this invention come in a size of 24separate wells (with inserts) per plate. Alternatively, multi-wellplates with a common, as opposed to separate, media reservoir can beused. The plates are typically wrapped with Nescofilm® or Parafilm™, orleft unwrapped and stored in incubators (27° C.) in the same manner thatis done with solid medium selection. The fact that the wells areseparated from each other is particularly beneficial because bacterialcontamination does not spread to other explants, as could be the case incommon medium reservoirs, felt liquid selection, or solid mediumselection.

The vector pMON 68410 was used for transformation and contained thenptII gene as a selectable marker and gfp as a scorable marker. Callustissue of LH244 was used as an explant. Transformation steps were thesame as disclosed in Example 8. Experiments were also done in the Costarmulti-well plates with and without different matrices for providingsupport to the explant in liquid medium. Glass beads, silicon beads,felt, foam, and solid media with additional liquid medium were used.Control treatments were: culture on solid medium in Petri plates (trt#1; Table 1) and only liquid medium in multi-well plates (trt #2; Table1). The different matrices were used in four experiments by fourdifferent people. All treatments yielded transgenic plants. However, useof silicon bead matrix and the small felts were at par with the controltreatment of culture on solid medium in Petri plates.

TABLE 1 Use of multi-well plates with and without matrices for producingtransgenic corn plants from callus explants. # of callus pieces for TrtMatrix/ each ex- % TF # Medium periment Exp. 1 Exp. 2 Exp. 3 Exp. 4Total 1 Gel/solid 48 39.6 50.0 50.0 25.0 41.1 in Petri Plate 2 None/ 244.2 25.0 16.7 8.3 13.5 Liquid 3 Glass 24 25.0 12.5 33.3 16.7 21.9 Beads/liquid 4 Silicon 24 33.3 25.0 29.2 50.0 34.4 Beads/ liquid 5 Small 2441.7 25.0 54.2 29.2 37.5 Felt/liquid 6 Foam 24 0.0 16.7 0.0 4.2 5.2 7Solid 24 16.7 16.7 12.5 4.2 12.5 medium with additional liquid medium

Table 2 shows that multiple-well system with (trt#3 to 5) and without(trt#6) matrices can also be used for producing transgenic corn plantsutilizing immature embryos as explants and glyphosate as a selectiveagent. The vector pMON 92690 for this study contained cp4 and gus genes.Transfer of media in multi-wells was done on a weekly basis.

TABLE 2 Use of multi-well plates with and without matrices for producingtransgenic corn plants from embryo explants. # of Trt # Container MediumMatrix embryos % TF 1 Petri plate Solid Gel 40 60.0% 2 Petri plateLiquid Felts 40 45.0% 3 Multi-well Liquid Silicon Beads 40 25.0% 4Multi-well Liquid Glass Beads 40 15.0% 5 Multi-well Liquid Small Felt 4037.5% 6 Multi-well Liquid None 48 2.1%

Example 5 Use of a Single Petri Dish with Solid Medium Followed byLiquid Medium

Another example of a single-container system is the use of a singlePetri dish with solid medium and subsequently adding liquid medium tofacilitate selection and regeneration in the single plate withoutreplacing the old medium. Corn callus was obtained and inoculated asdescribed in Example 7.

Callus was then co-cultured in a 25×100 deep Petri-dish containing oneWhatman filter paper and 100 μL of ½ MSPL medium at 23° C. After 3 days,the callus was transferred to solid medium 850QRT (4.33 g/L MS salts,0.5 mg/L thiamine HCl, 0.5 mg/L pyridoxine HCl, 0.5 mg/L nicotinic acid,100 mg/L myo-inositol, 0.5 g/L casein hydrolysate, 1.38 g/L proline, 30g/L sucrose, 0.5 mg/L 2,4-D, 10 μg/L BAP, 20 μM AgNO3, 500 mg/Lcarbenicillin, 100 mg/L paromomycin, pH 5.8, 6 g/L agar).

After 14 days, 7-10 mL of fresh 850QRTT medium (850QRT without the agarand with 200 mg/L paromomycin) was added and plates were incubated inlow light. After an additional 14 days, 7-10 mL of fresh 850RT medium(4.33 g/L MS salts, 0.5 mg/L thiamine HCl, 0.5 mg/L pyridoxine HCl, 0.5mg/L nicotinic acid, 100 mg/L myo-inositol, 0.5 g/L casein hydrolysate,1.38 g/L proline, 30 g/L sucrose, 0.5 mg/L, 10 μg/L BAP, 500 mg/Lcarbenicillin, 100 mg/L paromomycin, pH 5.8) was added and plates werefurther incubated in low light.

After another 14 days, the regenerated shoots were transferred to 632ATmedium (4.33 g/L MS salts, 0.5 mg/L thiamine HCl, 0.5 mg/L pyridoxineHCl, 0.5 mg/L nicotinic acid, 50 mg/L myo-inositol, 60 g/L sucrose, 250mg/L carbenicillin, 100 mg/L paromomycin, pH 5.8, 6 g/L agar).Regenerated plants were then transferred to phytatrays containing thesame medium after 14 days, and then transferred to soil in 2 weeks.

Plants selected and regenerated in solid media produced about 46% usabletransformed plants compared to 47% usable transformed plants from thecontrol method using liquid medium on a felt support.

Example 6 Agrobacterium-Mediated Transformation of Maize Using a NovelSuspension Culture Method

It is known in the art that the generation of transformable cellsuspension culture is time consuming and often results in generation ofnon-embryogenic cells. This example describes a highly reproduciblemethod (referred to here as “short suspension culture” or “SSC”) forproducing a rapid suspension culture that is highly embryogenic and verycompetent for Agrobacterium-mediated gene transformation. SSC as anexplant combines the desirable characteristics of callus (embryogenicand easy to use), and of suspension cultures (uniform, highlyregenerable, and amendable to high throughput production). In thisnon-limiting example, neomycin phosphotransferase II (nptII) wasemployed as a selectable marker and a standard binary vector system wasused for efficient selection and regeneration of Agrobacterium-mediatedstable transgenic events in maize.

Agrobacterium strain, plasmid, and culture. Disarmed Agrobacteriumtumefaciens EHA 101 harboring the binary vector pMON25457 was used inthis experiment. pMON25457 contained selectable (nptII) and reporter(uidA) genes, each driven by an enhanced 35S promoter (“e35S”) andfollowed by an untranslated hsp70 intron. The uidA gene has anadditional intron within its coding sequence to minimize bacterialexpression. Plasmids were introduced into the Agrobacterium strain byelectroporation with a Bio-Rad Gene Pulser operated at 2.5 kV and 400Ohms. Transformed colonies were selected on solid Luria-Bertani (LB)medium (Sambrook and Russell, 2001) containing 100 mg/L each ofkanamycin and gentamycin.

Induction and growth of Agrobacterium. Agrobacterium cells used fortransformation were pre-induced with acetosyringone (200 μM) and glucose(2%) in AB-based induction medium (0.1M MES, 0.5 mM NaH₂PO₄, 2% glucose,1 g/L NH₄Cl, 300 mg/L MgSO₄.7H₂O, 150 mg/L KCl, 10 mg/L anhydrous CaCl₂,2.5 mg/L FeSO₄.7H₂O, pH adjusted to 5.4 with NaOH).

A general procedure for inducing Agrobacterium cells follows. A loopfulof bacterial colonies was picked from a fresh plate and grown at 28° C.in 50 mL LB medium containing appropriate antibiotics. The opticaldensity at 660 nm of the bacterial culture is after about 15 to 24 hoursculturing was about 1.4. A 10-mL aliquot of the culture was transferredinto 50 mL fresh LB medium containing appropriate antibiotics and grownfor an additional 6 to 8 hours to an optical density at 660 nm of about1.2. The Agrobacterium cells were centrifuged at 4° C. for 10 min at3250×g. The resulting pellet was resuspended in the induction medium toa final optical density at 660 nm of about 0.2 and incubated at 28° C.for about 12 to 15 hours. Prior to use for transformation, theAgrobacterium cells were centrifuged at 4° C. for 10 min at 3250×g.After decanting the supernatant, the pellet was resuspended in ½ MSVImedium (2.2 g/L MS salt (Gibco), 1 mL/L of 1000× stock MS vitamins, 115mg/L proline, 10 g/L glucose, 20 g/L sucrose, pH 5.4, filter-sterilized)and supplemented with 200 μM acetosyringone. At least 100 mL of ½ MS VImedium supplemented with 200 μM acetosyringone was used for every 1 LAgrobacterium suspension. The resuspended cells were aliquoted intosmaller volumes, centrifuged at 4 degrees C. for 10 min at 3250×g, thesupernatant discarded and the pellets stored in ice until use (up to 4hours). Pellets were resuspended to a desired optical density with ½ MSVI medium supplemented with 200 μM acetosyringone, with a suspension ofabout 109 cells/mL giving an optical density at 660 nm of about 0.2.

Growth of stock plants and callus formation. Maize Hi-II and FBLLgenotypes were grown in the greenhouse at 16-h day length. Crossesinvolving these two genotypes were made and immature embryos wereexcised onto a modified N6 medium (Chu et al., 1976) supplemented with1.0 mg/L 2,4-D,1 mg/L thiamine HCl, 0.5 mg/L nicotinic acid, 0.91 g/LL-asparagine monohydrate, 100 mg/L myo-inositol, 0.5 g/L MES, 1.6 g/LMgCl₂.6H₂O, 100 mg/L casein hydrolysate, 0.69 g/L proline, and 20 g/Lsucrose; the modified N6 medium was solidified with 2 g/L Gelgro(catalogue number 150180, ICN Biomedicals) and medium pH was adjusted topH 5.8 with KOH (pre-autoclave). The same growth conditions were usedfor the elite genotype RBDQ2. The FBLL x Hi-II hybrid and RBDQ2 calluslines were sub-cultured at 2-week intervals and maintained at 28° C. inthe dark for up to four months on the same medium. This subcultureprotocol also worked with the Hi-II and FBLL-MAB lines.

Short suspension culture (SSC) formation. A general protocol forgenerating SSC from calli follows. To initiate SSC formation, about 2 gof calli, 2 weeks post sub-culture, were transferred to a 250-mL baffledflask containing 80 mL MS Fromm medium supplemented with 2.0 mg/L 2,4-D,20 g/L sucrose, 150 mg/L L-asparagine, 100 mg/L myo-inositol, 1 mL MSFromm 1000× vitamin stock containing 650 mg/L nicotinic acid, 125 mg/Lpyridoxine HCl, 125 mg/L thiamine HCl, and 125 mg/L calcium pantothenate(medium pH adjusted to 5.8 with KOH). The SSC were generated on agyratory shaker set to 170 rpm and 28 degrees C. During each subsequenttransfer, tissues were allowed to briefly settle to the bottom of theflasks in order to remove undesirable cell types (for example, cellsthat were elongated, thick-walled, low in cytoplasmic content, ornon-dividing), before the suspension was transferred with a wide-mouthFalcon™ sterile disposable pipette. On day 1 post initiation, the tissuefrom the flask were transferred to a fresh flask containing 80 mLmodified MS medium and grown for two more days; this transfer step wasrepeated at day 3 post initiation. On day 5 post initiation, the tissuewas equally divided between two flasks, giving about 2.5 mL packed cellvolume (PCV) per flask. Thereafter, the medium was replaced every 3days. By day 14 post initiation, the resulting packed cell volume ineach flask was about 6 mL (approximately 4 g per flask, or about afour-fold increase in total PCV over 2 weeks). These cells (“SSC”) weretransferred to fresh medium on day 14 post initiation, andtransformation was initiated on day 15 post initiation. The relativelyfrequent change of medium throughout this SSC procedure permitted rapidtissue proliferation. An additional advantage of the SSC procedure wasthat no visual selection of tissues was required at each transfer step,making this system both practical and reproducible.

Inoculation and co-culture. Three flasks containing a combined total ofabout 18 mL PCV were used in a transformation study of FBLL x Hi-IIhybrid SSC explants. Pilot experiments were conducted to enhanceparameters involved in the various transformation steps. A modifiedco-culture technique was used as described by Cheng et al. (2003), whichis incorporated by reference herein. A desiccation step was employedpost Agrobacterium infection, which was found to greatly increase T-DNAtransfer as well as to increase recovery of transgenic events.

FBLL x Hi-II hybrid SSC tissue from each flask along with some liquidmedium was equally divided and transferred to two wells of a six-welltissue culture dish (Corning CoStar™, non-treated, part number 9088),or, alternatively, to a 20×60 mm Petri dish The liquid medium wasremoved from each well or dish. Five mL of Agrobacterium suspension(OD660 nm ˜0.5) in ½ MS VI medium supplemented with 200 μMacetosyringone was added to each well or dish, followed by a 1-hinoculation period. At the end of the inoculation period, most of theAgrobacterium suspension was gently removed and the inoculated SSC cellswashed with 5 mL ½ MS VI medium supplemented with 200 μM acetosyringone.Cells from each well or dish were transferred to a Petri dish containing3 layers of sterile filter paper to absorb excess liquid from the cells,and then divided equally and transferred to two 60×20 mm Petri dishes,each containing a piece of 5.5 cm diameter sterile filter paper(Baxter). A total of 12 filter paper-containing co-culture dishes werethus obtained (4 from each original flask). The transferred cells werearranged in 6 to 8 clumps on the filter paper. One day co-cultivationunder desiccation was performed using a modified protocol of Cheng etal. (2003), which is herein incorporated by reference in its entirety.One hundred μL ½ MS VI medium supplemented with 200 μM acetosyringonewas added to each filter paper, and the plates were wrapped withParafilm™ and incubated in the dark at 23 degrees C. Transformationfrequency was calculated as number of independent events regenerated permL PCV.

Transformation experiments were similarly conducted with RBDQ2 SSCexplants, using three replicate packed cell volumes (4.5 mL each). Thetransformation procedure was generally the same as that used with theFBLL x Hi-II hybrid SSC explants, except that, for one of the two wellsper flask, ½ MS VI medium supplemented with 200 μM acetosyringone andcontaining 20 μM AgNO₃ was used during the inoculation and wash steps.

Selection and regeneration of transgenic plants. The protocol fortransformation of SSC with nptII using paromomycin selection involved asimple filter paper support for transfer of explants during selection,which minimized labor, ensured rapid elimination of Agrobacterium,increased growth rate of the tissue, and resulted in faster selection.

This procedure was performed on FBLL x Hi-II hybrid SSC as follows. Onday 0 of transformation (that is at the end of the co-culture step), awashing step was carried out with 5 mL MS Fromm medium supplemented with1000 mg/L carbenicillin, 100 mg/L ticarcillin, 100 mg/L vancomycin, and40 mM AgNO₃ that was added directly to each of the co-culture plates,and cell clumps gently tapped using a sterile spatula to ensuresubmersion. The wash medium was removed from the plates, and the filterpapers carrying the cells were transferred to Petri dishes containingfresh solid Duncan “D” medium containing 3.0 mg/L 2,4-D supplementedwith 1000 mg/L carbenicillin, 100 mg/L ticarcillin, and 40 micromolarAgNO₃ as a delay medium. Duncan “D” medium contains basal salts andvitamins of the “D” medium as described by Duncan et al. (1985), whichis incorporated by reference herein; generally 500 mL of a 2× stock ofthis medium was prepared and added to 500 mL of autoclaved steriledistilled water containing 6 g of Phytagar. Cells were maintained indelay medium for 6 days. On day 6 of transformation, a first selectionstep was performed with the filter papers carrying the cells transferredto Petri dishes containing Duncan “D” medium with 1.5 mg/L 2,4-Dsupplemented with 750 mg/L carbenicillin, 100 mg/L ticarcillin, 40micromolar AgNO3, and 50 mg/L paromomycin as a first selection medium.Two weeks after the end of the co-culture step, a second selection stepwas performed with the filter papers carrying the cells transferred toPetri dishes containing Duncan “D” medium with 1.5 mg/L 2,4-Dsupplemented with 750 mg/L carbenicillin, 100 mg/L ticarcillin, 40micromolar AgNO3, and 100 mg/L paromomycin as a second selection medium.Three weeks after the end of the co-culture step, a third selection stepwas performed with pea-sized clumps of tissue removed from each filterpaper and plated onto a solid Duncan “D” medium with 1.5 mg/L 2,4-Dsupplemented with 750 mg/L carbenicillin, 100 mg/L ticarcillin, and 100mg/L paromomycin as a third selection medium. There were about 4 to 6resulting clumps of cells plated from each filter paper, and each cellclump was treated individually henceforth in the experiments. One platedclump per filter paper was histochemically assayed for transgenic sectorsize, which was found to be about 1 to 2 mm in diameter. Four weeksafter the end of the co-culture step, a fourth selection step wasperformed with each clump transferred to Petri dishes containing Duncan“D” medium supplemented with 1.5 mg/L 2,4-D supplemented with 750 mg/Lcarbenicillin, 100 mg/L ticarcillin, and 100 mg/L paromomycin. At theend of this fourth selection cycle, a total of 232 paromomycin-resistantFBLL x Hi-II hybrid lines were obtained. Seven weeks after the end ofthe co-culture step, a selection and pre-regeneration step is wascarried out with the resistant calli transferred to deep Petri dishes(20×100 mm) containing 3.52 mg/L 6-BAP, 500 mg/L carbenicillin, and 100mg/L paromomycin for one week. Finally, regenerating tissues weretransferred to an MS regeneration medium for two weeks in Petri dishes,followed by transfer to a Phytatray for an additional three weeks. MSregeneration medium consisted of modified MS medium supplemented with 10g/L glucose, 20 g/L sucrose, 100 mg/L myo-inositol, 150 mg/LL-asparagine, and 1 mL/L MS Fromm 1000× vitamin stock containing 650mg/L nicotinic acid, 125 mg/L pyridoxine HCl, 125 mg/L thiamine HCl, and125 mg/L calcium pantothenate, and 6 g/L Phytagar; medium pH wasadjusted to 5.8 with KOH pre-autoclave, and 250 mg/L carbenicillin and100 mg/L paromomycin were added post-autoclave. Thirteen weeks after theend of the co-culture step, the resulting plantlets were transferred tosoil.

Progeny analysis of transgenic plants. UidA activity was assayed atvarious stages of selection and transformation, following thehistochemical procedure described by Jefferson (1987), which isincorporated by reference herein, at various stages of transformationand plant growth. Detection and copy number analysis of nptII wasperformed with INVADER® assays (Third Wave Technologies, Madison, Wis.).At least under the experimental conditions described above, a delay stepfollowed by a step-wise increase in selection pressure was found to benecessary to recover transgenic sectors from SSC explants. In general,Agrobacterium-mediated transformation competence of maize cells wasfound to be improved by subjecting callus to at least a short liquidphase prior to initiating transformation. For example, FBLL x Hi-IIhybrid SSC explants were found to be highly competent to T-DNA transferbetween about 5 days to about 14 days post initiation of SSC with 80% ofthe tissues expressing gus at 3 days post transformation. Older (2month) SSC cultures were also found to be competent to transformation,with about 20% of the explants exhibiting histochemical staining foruidA activity 3 days post transformation. In contrast, FBLL x Hi-IIhybrid calli maintained on 211 modified N6 medium were found to bepoorly competent, with less than 20% of the explants exhibitinghistochemical staining for UidA activity 3 days post transformation.

In the case of the FBLL x Hi-II hybrid genotype, a total of 232paromomycin-resistant lines were obtained from the original threeflasks. Of 76 resistant callus lines tested, 56 (about 74%) were foundto express uidA. A sub-set of 35 paromomycin-resistant lines wereregenerated; of these, 24 of 25 events that were regenerated into plantswere found to contain uidA. Southern blots and histochemical assays ofplants arising out of several SSC experiments confirmed integration ofthe transgene in the R1 generation, and inheritance of the transgenes byR0 and R1 generations. In the case of the RBDQ2 genotype, 3 replicateflasks (each containing about 4 mL PCV) yielded a total of 37paromomycin-resistant, uidA-positive putative events. Transformationfrequency was underestimated because some tissues were sacrificed forUidA histochemical assays. In this genotype, use of AgNO₃ duringinoculation or washing did not enhance transformation frequency,suggesting that suppression of Agrobacterium growth by desiccation wassufficient to minimize Agrobacterium toxicity. A total of 29 putativeparomomycin-resistant calli were generated, out of which 18 putativeevents were transferred to soil. Seventeen of the 18 events were foundto be uidA-positive by histochemical assays of leaf tissue. Thus,transformation frequency of RBDQ2 SSC (based on the putative events) wasestimated to be approximately 1.4 event/1 ml PCV (17 events/12 mL PCV).

This SSC procedure was thus capable of rapidly establishing regenerabletype II suspension cultures, suitable for Agrobacterium-mediated stabletransformation, from both the FBLL x Hi-II hybrid and the proprietaryelite RBDQ2 genotypes. SSC explants may be especially useful in thedevelopment of a high-throughput gene evaluation system.

Example 7 Preparation of Agrobacterium for Transformation by DirectInoculation of a Frozen Glycerol Stock of Agrobacterium into anInduction Medium

This is a non-limiting example of one method of preparing Agrobacteriumfor transformation. More specifically, this example describes directinoculation of a frozen glycerol stock of Agrobacterium into a virinduction broth.

The transfer of T-DNA into plant cells using the Ti plasmid requiresactivation of the vir genes encoding the proteins VirA and VirG. Thesignals for VirA activation include, for example, acidic pH, phenoliccompounds (for example, acetosyringone), and certain sugars that actsynergistically with phenolic compounds. Conventionally, pre-inductionof vir genes involve growing Agrobacterium cells (usually from a frozenstock) in a growth medium for a varying period of time, measuringAgrobacterium density, adjusting to a desired density, growing in ABminimal induction medium or other suitable medium for induction,spinning and washing the cells, and finally adjusting the Agrobacteriumculture to a desired density prior to inoculation. The numerous stepsinvolved require considerable time and effort, and provide opportunitiesfor undesired variability in experiments.

Disclosed here is a rapid, single-step Agrobacterium direct inductionprocedure that reduces time, effort, and variability in transformationexperiments. The reduction in number of steps required for induction isalso advantageous for quality control purposes and reduction ofergonomic burden.

In the non-limiting embodiments described herein, the vector pMON 70801carrying a gene (cp4) for glyphosate resistance (see U.S. Pat. No.5,633,435, which is incorporated by reference in its entirety herein)was used; however, many other Agrobacterium vectors could be used, as isknown in the art. Optical densities (OD) were measured at 660nanometers. Additional suitable procedures, including descriptions ofmedia and reagents, for transformation of plants using glyphosateselection, have been disclosed, for example, in U.S. Patent Appln.Publn. 2004/0244075 to Cai et al., which is incorporated by reference inits entirety herein.

Direct Induction with Modified MS Induction Medium. In a non-limitingembodiment, the procedure can involve pre-induction of a frozenAgrobacterium glycerol stock in an MS induction medium containingnecessary vir induction components. After pre-induction, theAgrobacterium cells are used directly in transformation experiments.

Agrobacterium was grown from a frozen stock made with MS medium (½MSsalts, 100 μM MES, 2% glucose with 25% (v/v) glycerol). The frozen stockmade with MS salts was resuspended in a modified MS induction brothcontaining acetosyringone (½MS salts, 100 μM MES, 2% glucosesupplemented with appropriate antibiotics (Spec/Strep 50 μg/milliliter,Kanamycin 50 μg/milliliter, and Chloramphenicol 25micrograms/milliliter, and 200 μM acetosyringone). A total of 5 hoursinduction was followed with an initial OD of 0.4. For induction, oneflask (250-mL baffled flask) with 50 milliliters of induction medium wasused. At the end of the induction Agrobacterium suspension was useddirectly for inoculation and was supplemented with 20 μM AgNO₃ justprior to inoculation.

The above-described modified MS direct induction protocol was comparedwith a conventional protocol involving the following steps:Agrobacterium was grown from a frozen stock for 8 hours in LB liquidwith appropriate antibiotics (Spec/Strep 100 micrograms/milliliter,kanamycin100 micrograms/milliliter, and chloramphenicol 25micrograms/milliliter). Agrobacterium was spun down and resuspended inan AB minimal induction broth with appropriate antibiotics (spec/Strep50 micrograms/milliliter, kanamycin 50 micrograms/milliliter, andchloramphenicol 25 micrograms/milliliter), and 200 micromolaracetosyringone. A total of 13 hours induction was followed with aninitial OD of 0.2. For induction one flask (1-liter baffled flask)containing 300 milliliters of induction medium was used. At the end ofinduction Agrobacterium was spun down and resuspended with ½MSVIsupplemented with 200 micromolar acetosyringone to an OD of 0.4. AgNO₃was added to a final concentration of 20 micromolar just prior toinoculation.

Explants for this study were calli derived from a near-elite corn linesub-cultured on fresh medium 8 days prior to initiating transformationon N6-based 201 medium. Inoculation was performed in 25×100 millimeterdeep dishes. About 6 grams of calli were transferred to a deep dishcontaining 50 milliliters of ½ MS VI (one dish per treatment). Explantswere washed and all liquid was removed. Twenty milliliters ofAgrobacterium suspension (OD 0.4) supplemented with acetosyringone(final concentration 200 micromolar) and AgNO₃ (final concentration of20 micromolar), and a 1 hour inoculation was performed. Agrobacteriumsuspension was removed after inoculation and the explants were washedwith 15 milliliters ½ MSVI supplemented to concentration of 20micromolar AgNO₃ and 200 micromolar acetosyringone. Calli from eachinoculation dish were transferred to a deep dish containing 10 filterpapers (7.5 millimeters; Baxter catalogue number 28313-046) to removethe excess liquid. About 0.5 is gram (wet weight) or about 2.5 grams(drained weight) of callus was transferred to a single co-culture plate(25×60 millimeters) containing a single filter paper (5.5 millimeters;Baxter catalogue number 28297-868). Explants were arranged in fourgroups towards the edge of the co-culture plate. Co-culture wasperformed with or without desiccation (1 milliliter of ½ MSVI was added)at 23° C. for 18 hours. For each treatment, that is conventionalinduction or modified MS induction, seven replicates (4 withoutdesiccation, 3 with desiccation) were used. No statistically significantdifference was observed between the two induction protocols. Themodified MS direct induction protocol was shown to produce about thesame number of putative events as did the lengthier conventionalprotocol.

Direct induction with AB induction medium. In another non-limitingembodiment, the procedure involved pre-induction of a frozenAgrobacterium glycerol stock in an AB induction medium containingnecessary vir induction components. After pre-induction, theAgrobacterium cells were adjusted to the desired density and useddirectly in transformation experiments. Agrobacterium was grown from aregular LB frozen stock with an Agrobacterium optical density (OD) of3.0 measured at 660 nanometers (final concentration).

An Agrobacterium culture was prepared and split into two portions. Oneportion was grown for an additional 4.5 hours to OD ˜0.6 in 36milliliters of 2XYT medium, centrifuged, resuspended at OD ˜3.0 in ABminimal medium containing 20% glycerol, and frozen for use in a directinduction protocol. For induction, 5 milliliters of the frozen stock wasthawed, added to 70 milliliters AB minimal medium containingacetosyringone, and induced overnight.

The second Agrobacterium culture portion was streaked onto plates andincubated 3 days; a first seed culture prepared from these plates wasgrown overnight. This was followed by a second seed culture preparedfrom the first seed culture, grown for a day and induced overnight at OD˜0.2 in AB minimal medium containing acetosyringone.

The two induced Agrobacterium preparations were used to inoculate maizeembryos in parallel experiments. Maize embryos from half of each earwere excised into each of the Agrobacterium preparations (OD ˜1.0) for15 minutes, and then allowed to sit for 5 minutes. The embryos wereremoved and placed onto co-culture plates. Using the is conventionalprotocol, a total of 883 embryos were inoculated, resulting in a totalof 174 (20%) plants to soil and an average transformation frequency of17% (standard deviation=11.4). Using the direct induction protocol, atotal of 814 embryos were inoculated, resulting in a total of 153 (19%)plants to soil and an average transformation frequency of 19% (standarddeviation =11.6). Copy number of the cp4 transgene was found to besimilar between treatments.

Example 8 Methods for Preparing Corn Callus for Sub-Culturing byMechanical Means

One of the greatest ergonomic burden and time-consuming processes of acorn transformation production system is the establishment andmaintenance of embryogenic callus for Agrobacterium mediatedtransformation which requires manual breaking of callus during thesub-culturing step. Furthermore, during the transformation process, manycells are transformed in a single immature embryo or a single piece ofcallus. However, current selection and regeneration practices treat eachpopulation of transformed cells in a single immature embryo or piece ofcallus as a single event, regenerating usually one plant per piece oftissue.

In order to relieve the ergonomic burden created by manual pinching byforceps of callus, especially type I callus, several mechanical meanssuch as coffee grinders, baby food mills, peppercorn grinders, herbgrinders, garlic cutters, and garnish knives were tested as a means forbreaking or cutting callus for sub-culturing. Among these means, herbgrinder and garnish knife were found to be most suitable for breaking orcutting callus and in producing callus pieces suitable for subcultureand transformation.

Use of mechanical means for preparing corn callus for sub-culturing canalso be used to separate many transformed cells within a given unit oftissue by cutting or breaking up the tissue into smaller units ofindividually transformed cells from which a transformed plant may beregenerated thus obtaining more transgenic plants from the same unit oftissue.

The callus for this example was derived by culturing immature cornembryos on is callus medium 1074 (Table 3), that were isolated fromdeveloping kernels about 10 days after pollination. Five to nine weekold callus was treated with an herb grinder and contacted withAgrobacterium containing a vector carrying nptII gene for selection forup to 60 minutes. The inoculated callus was blot dried andco-culture/desiccated for 2-3 days at 23° C. in dark. The callus tissuewas then transferred to felt pieces in liquid selection medium 1086(Table 3) containing about 50 mg/L of paromomycin and cultured for up to30 days at 27-28° C. in dark to select for the transformed tissuecontaining the nptII selectable marker gene. The transformed tissue wasthen regenerated into plants by culturing the tissue surviving selectionin shooting medium 1087 (Table 3) containing BAP to induce shoots for5-7 days at 27-28° C. in 16-h light. The growing tissue was incubated inmedium 1067 (Table 3) for further 2-3 weeks to induce roots. Healthyshoots with or without roots were transferred to Phytatrays containingmedium 1067 and subsequently to soil for growing.

TABLE 3 Media compositions used. Media Components/L (Supplier) 1074 10861087 1067 MS Basal Salts 4.33 g 4.33 g 4.33 g 4.33 (Phytotech) MSVitamins (100X) 10 mL 10 mL 0 0 (Phytotech) MS Fromm Vitamins 0 0 1 mL 1mL (1000X)* BAP (Sigma) 0 0 3.5 mg 0 Thiamine HCL (Sigma) 0.5 mg 0.5 mg0 0 2,4-D (Phytotech) 0.5 mg 0.5 mg 0 0 Sucrose (Phytotech) 30 g 30 g 30g 0 Glucose (Phytotech) 0 0 0 10 g Maltose (Phytotech) 0 0 0 20 gProline (Sigma) 1.38 g 1.38 g 1.38 g 0 Casamino Acids 0.5 g 0.5 g 0.5 g0 (Difco) Asparagine 0 0 0 0.15 g monohydrate (Sigma) Myo-inositol(Sigma) 0 0 0 0.1 g Phytagel (Sigma) 3 g 0 3 g 0 Phytagar (Gibco) 0 0 06 g Carbenicillin 0 500 mg 250 mg 250 mg (Phytotech) Picloram (Sigma)2.2 mg 2.2 mg 0 0 Paromomycin 0 100 mg 100 mg 100 mg (Phytotech) SilverNitrate (Sigma) 3.4 mg 3.4 mg 0 0 pH 5.8 5.8 5.8 5.8 comprising 1250mg/L nicotinic acid (Sigma), 250 mg/L pyridoxine HCl (Sigma), 250 mg/Lthiamine HCl (Sigma), and 250 mg/L calcium pantothenate (Sigma).

The results indicate that the herb grinder can be successfully used tobreak callus into pieces suitable for sub-culturing and transformation(Table 4).

TABLE 4 Use of herb grinder for breaking callus pieces for sub-culturingand transformation. No. of callus No. of Treatment & Grams of pieces putto plants TF Expt Description callus used selection produced (%) 1 1.Hand excised, 2.5 192 53 27.6 hand subcultured 2. Hand excised, 2.5 18539 21.1 mechanical subcultured 2 1. Hand excised, 1.5 144 9 6.3 handsubcultured 2. Hand excised, 1.5 116 17 14.7 mechanical subcultured

A garnish knife (Pickle Slicer, #4428 from International Culinary,Mystic, Conn.) was also used a means for cutting callus for reducing theergonomic burden of hand-subculture methods. The garnish knife consistedof 8 parallel blades attached to the end of a plastic handle. The knifewas sterilized with a mild bleach soak, followed by an ethanol soak, andthen left to dry for approximately 30 minutes. While the knife wasdrying, 4-week old callus (hand-sub-cultured at 2 weeks) was placed in a150×15 mm Petri dish, and divided into 3 to 4 smaller piles within thedish, each containing approximately 40-60 callus pieces. Working withone pile at a time, the knife was used to cut through the callus. With asterile implement, the callus was pushed out of the knife that hadcollected between the blades onto the plate, where it was cut a secondtime. Other callus piles were treated the same way. The cut callus wasused for sub-culturing and transformation.

Data from several garnish knife experiments showed a transformationfrequency is of 13.16%. In additional tests, the transformationfrequency of hand subcultured callus of 21.5% was comparable to callusprepared by garnish knife (18.9%). In yet another 74 experiments usinggarnish knife as a means for cutting callus by 5 different users, theaverage transformation frequencies across all users was 19.4% indicatingutility of this method.

Example 9 Methods for Inoculating Explants with Agrobacterium forTransformation

The current methods for Agrobacterium-mediated transformation, forexample, of corn immature embryos uses many steps involving excising oneembryo at a time from the kernels, incubating them in Agrobacterium cellsuspension, removing Agrobacterium suspension, transferring the embryosto a co-culture plate for infection to occur, orienting the embryos andplacing them with scutellum side up and finally transferring the embryosto a selection or delay medium for further manipulations such asselection and regeneration. Many of these steps are ergonomicallyunfriendly and also raise the possibility of damaging excised embryosand increased incidence of Agrobacterium-related cell death therebyresulting into reduced transformation frequency. Therefore, there is aneed in the art of corn transformation to simplify inoculation step,such that it is ergonomically friendly and results in highertransformation frequency.

The inventors have now simplified the inoculation step by reducing thetotal number of steps by inoculating transformable explants, forexample, immature embryo, by either 1) dipping a spatula inAgrobacterium suspension prior to excising the embryo with spatula; 2)by soaking the corn ear with Agrobacterium suspension before excisingthe embryo; or 3) by combing the two steps for example, by soaking thecorn ear with the Agrobacterium suspension followed by excising theembryos by a spatula dipped in Agrobacterium suspension.

In general, the following transformation method was used. Immature cornembryos from a recipient corn line were dissected from developingkernels about 10 days after pollination and inoculated by eitherincubation, dipped spatula (18.0%), or soaking the ear withAgrobacterium containing the construct. The inoculated embryos wereco-cultured on co-culture medium 1514 (Table 5) for about 24 hr at 23°C. in dark. The embryos were then transferred for selection ontoselection medium 1278 (Table 5) containing 0.1 mM glyphosate to selectfor the transformed tissue containing the cp4 selectable marker gene and500 mg/L Carbenicillin to inhibit Agrobacterium growth by incubating for2-3 weeks at 27° C. in dark. The transformed corn tissue was thenregenerated into plants by transferring transformed callus pieces ontothe first regeneration medium 1073 (Table 5) and grown for 5-7 days with16 hours light/8 hours dark photoperiod and 27° C. temperature. Thetissue was then transferred onto the second regeneration medium 1071(Table 5) for approximately 2 weeks. Regenerated shoots were transferredto rooting medium 1084 (Table 5) in Phytatrays. The developing plantletswere then transferred to soil, hardened off in a growth chamber at 27°C., 80% humidity, and low light intensity for approximately 1 week andthen transferred to a greenhouse and grown under standard greenhouseconditions.

The Agrobacterium carrying plasmid pMON 92689 containing a modified CP4aroA gene for glyphosate selection (U.S. Pat. No. 5,627,061) was grownfor inoculation from a frozen stock made with MS induction medium asdescribed in Example 7. The kernels on the cob were sterilized with 80%ethanol and cut at the crown and then washed with 100 ml of ½ MSVI andapplied with 20 ml of Agrobacterium suspension. A spatula was dipped inAgrobacterium suspension and immature embryos were excised and plated onto the co-culture medium 1514 for 24 hrs at 23° C. in dark. Thesetreatments were is compared with a standard treatment wherein embryoswere excised one at a time and incubated with Agrobacterium suspensionfor up to 5 min. The embryo size was 1.5-1.8 mm. However, the method wasfound to work well with other embryos sizes as well, for example, embryosizes ranging from 1.4 to 2.2 mm. Transgenic events could be obtained bydirectly inoculating the explants with Agrobacterium while the explantswere still attached to the maternal tissue and by inoculating explantswhile they were being removed from the source. Following transformationfrequency were obtained for each treatment: inoculation by incubation(20.7%), inoculation by dipped spatula (18.0%), and inoculation bysoaking the ear (17.0%).

Other means of contacting explants with Agrobacterium and duration ofcontact may be anticipated by those skilled in the art. For example, aknife dipped in Agrobacterium suspension can be used for inoculation bycutting leaf or callus tissue into smaller pieces or a dipped needle maybe used to inoculate the plant tissue by inserting the needle into theplant tissue.

The method has been found to improve transformation frequency byallowing reduced tissue handling and reduced Agrobacterium-related celldeath. With 6 different constructs, the dipped spatula method producedan average TF of 14.3% over standard incubation method which produced aTF of 9.2%.

TABLE 5 Media compositions used. Media Components/L (Suppliers) 12331278 1514 1524 1073 1071 1084 MS Basal Salts 2.165 g 4.33 g 4.33 g 4.33g 4.33 g 4.33 g 2.165 g (Phytotech) MS Vitamins 10 mL 10 mL 10 mL 10 mL0 0 0 (100X) (Phytotech) MS Fromm 0 0 0 0 1 mL 1 mL 0 Vitamins (1000X)*BAP (Sigma) 0 0.01 mg 0 0 3.5 mg 0 0 Thiamine HCL 0.5 mg 0.5 mg 0.5 mg0.5 mg 0 0 0 (Sigma) 2,4-D (Phytotech) 3 mg 0.5 mg 0.5 mg 0.5 mg 0 0 0NAA (Sigma) 0 0 0 0 0 0 0.5 mg IBA (Sigma) 0 0 0 0 0 0 0.75 mg Sucrose(Phytotech) 20 g 30 g 30 g 30 g 30 g 0 20 g Glucose (Phytotech) 10 g 0 00 0 10 g 0 Maltose (Phytotech) 0 0 0 0 0 20 g 0 Proline (Sigma) 115 mg1.38 g 1.38 g 1.38 g 1.38 g 0 0 Casamino Acids 0 0.5 g 0.5 g 0.5 g 0.05g 0.5 0 (Difco) Asparagine 0 0 0 0 0 0.15 0 monohydrate (Sigma)Myo-inositol 0 0 0 0 0 0.1 g 0 (Sigma) Low EEO Agarose 5.5 g 0 0 0 0 0 0(Sigma) Phytagel (Sigma) 0 3 g 3 g 3 g 3 g 3 g 3 g Acetosyringone 200 uM0 0 0 0 0 0 (Aldrich) Carbenicillin 500 mg 500 mg 500 mg 500 mg 250 mg250 mg 0 (Phytotech) Glyphosate 0 0.1 mM 0.1 mM 0.1 mM 0.1 mM 0.1 mM 0.1mM (Gateway Chemical) Silver Nitrate 3.4 mg 3.4 mg 3.4 mg 3.4 mg 0 0 0(Sigma) pH 5.2 5.8 5.8 5.8 5.8 5.8 5.8 *Comprising 1250 mg/L nicotinicacid (Sigma), 250 mg/L pyridoxine HCl (Sigma), 250 mg/L thiamine HCl(Sigma), and 250 mg/L calcium pantothenate (Sigma). For liquid mediaPhytagel was excluded from the composition.

Example 10 Agrobacterium-Mediated Transformation of Corn by DoingInoculation, Co-Culture, and Selection in a Single Step

Normally Agrobacterium-mediated transformation methods involve 3distinct steps: inoculation of an explant with Agrobacterium,co-cultivation of inoculated explant on a medium absent of an antibioticto allow for survival of Agrobacterium and to enhance infection of theexplant, and selection of the transformed cell on a medium containing aselective agent such as kanamycin and glyphosate. There is always a needto reduce the number of steps required in a transformation method toimprove production efficiencies. The inventors have now provided amethod for Agrobacterium-mediated transformation of corn whereininoculation, co-culture and selection steps can be combined into asingle step by plating the inoculated explants directly onto a mediumthat contains selective agents for suppressing growth of Agrobacteriumand killing non-transformed explant cells thereby allowing inoculation,co-culture, and selection in a is single step, thus improvingtransformation production system efficiencies.

In general the transformation method disclosed in Example 9 was used.The Agrobacterium containing pMON65375 containing a modified CP4 aroAgene for glyphosate selection (U.S. Pat. No. 5,627,061) for inoculationwas prepared from a frozen stock made in AB minimal induction medium asdisclosed in Example 7.

The spatula was dipped in Agrobacterium suspension prior to excisingeach embryo and excised embryos were plated onto co-culture medium 1233and then on selection medium 1278 or plated directly onto the selectionmedium 1278. The results show that transformation can be done bycombining inoculation, co-culture and selection in a single step with avariety of Agrobacterium concentrations and embryo sizes.

In another embodiment, transformation was also achieved by firstculturing embryos on a selection medium and then applying Agrobacteriumsuspension immediately to the embryos rather than first applyingAgrobacterium and then culturing embryos on the selection medium.Transgenic plants could also be obtained by applying additionalAgrobacterium during co-culturing and selection.

Example 11 Selection of Transformed Cells on Sorbarods and Developmentof High-Throughput System

Suitable explants such as callus and immature embryos can be obtainedvia mechanical and manual excision means known in the art and can beinoculated with Agrobacterium containing a plasmid comprising any geneof interest known in the art. In one embodiment, the selection of atransformed cell is done on a felt piece placed in the liquid medium andregeneration is done on Sorbarod™ plug (Baumgartner Papiers SA,Crissier-Lausanne, Switzerland) placed in the liquid medium. In anotherembodiment, both selection and regeneration of the transformed cell aredone on a Sorbarod™ plug placed in the liquid medium.

The utility of these embodiments is demonstrated by several examples ofdata provided in Table 6. Compositions of media used are shown in Table5 except for liquid media Phytagel was excluded. Immature corn embryosfrom the recipient line were is dissected from developing kernels andinoculated with Agrobacterium containing binary vector pMON42073comprising a CP4 gene for glyphosate selection. The inoculated embryoswere co-cultured on co-culture medium 1514 (see Table 5 for composition)for about 24 hr at 23° C. in dark. The inoculated embryos were thentransferred to either a 5×5 cm square felt piece (Consumer ProductsEnterprises (CPE) Inc., Union, S.C.) or a Sorbarod plug (ILACON Limited,Kent, UK) placed in the liquid selection medium 1278 (see Table 5 forcomposition) containing 0.1 mM glyphosate to select for the transformedtissue. After this step, used selection liquid medium 1278 was removedand replaced with the liquid first regeneration medium 1073 (see Table 5for composition). The tissue was grown for 5-7 days with 16 hours oflight at 27° C. Normally, this step requires transferring of selectedtissue to solid first regeneration medium for further growth. Thismethod eliminates this step. The partially regenerated tissue on theliquid first regeneration medium was then transferred to a Sorbarod plugplaced in a liquid second regeneration medium (see Table 5 forcomposition) in a Sundae cup for full regeneration. This transfer stepwas eliminated if the inoculated embryos were cultured directly on theSorbarod plug to begin with. In such a case, the liquid firstregeneration medium was simply removed and replaced with the liquidsecond regeneration medium. The tissue was grown for about 2 weeks.Normally, this step requires transferring of the regenerating tissuefrom the first solid regeneration medium to the solid secondregeneration medium for further growth. This method eliminates thisstep. Fully regenerated smaller corn plantlets in the Sorbarod plug weretransferred to soil directly. Normally, this step requires transferringa fully regenerated smaller plantlet from the solid second regenerationmedium to the solid rooting medium 1084 (see Table 5 for composition)for further growth. This method eliminates this step. The developingplantlets were hardened off in a growth chamber at 27° C., 80% humidity,and low light intensity for about 1 week and then transferred to agreenhouse and grown under standard greenhouse conditions and tested forthe presence of CP4 gene. Table 6 shows the production of transgeniccorn using liquid culture in combination with felt and Sorbarod plug andby eliminating several transfer steps.

A high throughput transformation system for corn is developed in whichcontainers are manipulated by robotic arms on a freely configurable worktable that is include usable incubators and shakers in addition tostandard lab ware. Various liquid handling tools equipped with one ormore pipetting tips are used to provide the fresh medium and remove theexpensed medium. Work table, robotic arms, and the liquid handling toolsare controlled by software via a computer. Alternatively, liquid mediumfor selecting and regenerating the transformed cell can be provided tothe container via one or more tube connected to a medium storage vesseland removed via one or more tubes connected to a waste vessel. Theprovision and removal of the liquid medium is controlled manually ormechanically.

TABLE 6 Corn transformation frequency (TF) using liquid medium incombination with different matrices. Transformation frequency iscalculated as the percent of cultured embryos that produced transgenicplants. Culture conditions during # Plants Medium Matrix selection #Explants regenerated % TF Ear 1 Solid None 14-21 d, 27° C. dark 20 2 10Solid None 10-d, 30° C. dark; 7-d, 28° C. light 20 3 15 Liquid plug10-d, 30° C. dark; 7-d, 28° C. light 72 5 6.9 Liquid felt/plug 10-d, 30°C. dark; 7-d, 28° C. light 63 5 7.9 Ear 2 Solid None 14-21 d, 27° C.dark 20 2 10 Solid None 10-d, 30° C. dark; 7-d, 28° C. light 20 4 20Liquid plug 10-d, 30° C. dark; 7-d, 28° C. light 72 7 9.7 Liquidfelt/plug 10-d, 30° C. dark; 7-d, 28° C. light 63 8 12.7 Ear 3 SolidNone 14-21 d, 27° C. dark 30 6 20 Solid None 10-d, 30° C. dark; 7-d, 28°C. light 30 9 30 Liquid plug 10-d, 30° C. dark; 7-d, 28° C. light 70 710 Liquid felt/plug 10-d, 30° C. dark; 7-d, 28° C. light 63 8 12.7

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

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What is claimed:
 1. A method for inoculating a maize explant, comprisingpreparing an Agrobacterium cell suspension by culturing a frozenglycerol stock of Agrobacterium comprising a plant transformationconstruct directly into an induction medium containing a phenoliccompound for vir gene induction, and inoculating said maize explant withthe prepared Agrobacterium cell suspension, to produce an inoculatedmaize explant, wherein the explant is selected from the group consistingof: a mature embryo; an immature embryo; a meristem and callus tissue.2. The method of claim 1, wherein the induction medium comprises MSsalts.
 3. The method of claim 1, wherein the induction medium comprisesacetosyringone.
 4. The method of claim 1, wherein the induction mediumis AB medium.
 5. The method of claim 1, wherein the explant isinoculated by incubation in the cell suspension; or by contact with aspatula, knife, or needle dipped in the cell suspension.
 6. The methodof claim 1, wherein the explant is inoculated while attached to maternaltissue.
 7. The method of claim 1, further comprising co-culturing theinoculated explant.
 8. The method of claim 7, further comprisingregenerating a transgenic plant from the inoculated explant.